Despite initial expectations, adenovirus (Ad)- mediated gene therapy remains stymied by a lack of fundamental knowledge of host cell interactions. Nonetheless, Ad continues to serve as an important tool for uncovering key cellular and molecular processes. In previous studies, we discovered an integrin-signaling pathway that promotes Ad uptake into host cells. However, significant gaps remain in our understanding of further intracellular trafficking events. In particular, the mechanism by which Ad penetrates the early endosome remains poorly defined. Therefore, an experienced team of investigators will characterize the cellular and viral processes that facilitate Ad-mediated endosome disruption. The role of a key signaling molecule, protein kinase C, that promotes a late step in Ad entry via integrin avB5, will be investigated. Biochemical and biophysical studies will identify specific conformational changes in Ad particles required for membrane association and/or disruption. Improved cryo-electron microscopy and x-ray diffraction techniques will be used to reveal structural features of Ad particles as well as the conformational changes associated with membrane association. Finally, we will exploit structurally defined macromolecular platforms (i.e. icosahedral viruses) to investigate how the precise spatial orientation of integrin binding domains (RGD sequences) regulates virus entry or cell migration. These studies should provide a more comprehensive picture of Ad-host cell entry as well as reveal the underlying signaling events in virus internalization and cell migration. If successful, this new knowledge may also facilitate improvements in both viral and nonviral gene delivery systems.